Fragile X mental retardation protein FMRP binds mRNAs in the nucleus.
The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.
Kim
Miri
M
Dept. of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, Illinois 61801, USA.
Bellini
Michel
M
Ceman
Stephanie
S
D021581:Active Transport, Cell Nucleus; D000818:Animals; D000906:Antibodies; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D019556:COS Cells; D002452:Cell Count; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005453:Fluorescence; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017384:Sequence Deletion; D014981:Xenopus
D000906:Antibodies; D000952:Antigens, Polyomavirus Transforming; D051860:Fragile X Messenger Ribonucleoprotein 1; D049452:Green Fluorescent Proteins; D019913:Nuclear Localization Signals; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein